Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study, we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor, based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway, ribosome-related, and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes, with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.

SOX9 encodes an SRY-related transcription factor critical for chondrogenesis and sex determination among other processes. Loss-of-function variants cause campomelic dysplasia and Pierre Robin sequence, while both gain- and loss-of-function variants cause disorders of sex development. SOX9 has also been linked to scoliosis and cancers, but variants are undetermined. It is highly expressed in tooth progenitor cells, but its odontogenic roles remain elusive, and tooth defects are unreported in SOX9-related conditions. Here, we performed whole-exome sequencing for nine unrelated children with tooth eruption delay and no known syndromes and identified a 7-year-old girl heterozygous for a SOX9 p.Thr239Pro variant and a 10-year-old boy heterozygous for presumably adjacent p.Thr239Pro and p.Thr240Pro variants. These variants were de novo and rare in control populations. Both cases had primary tooth eruption delay. Additionally, the boy had mesiodens blocking permanent central upper incisor eruption, severe scoliosis, and mild craniofacial and appendicular skeleton abnormalities. p.Thr239 and p.Thr240 occupy variable and obligatory positions, respectively, in a cell division control protein 4 (Cdc4)/FBXW7-targeted phosphodegron motif (CPD) fully conserved in SOX9 vertebrate orthologs and SOX8 and SOX10 paralogs, but functionally uncharacterized in vivo. Structural modeling predicted p.Thr240Pro and p.Thr239Pro/p.Thr240Pro but not p.Thr239Pro to strongly reduce SOX9/FBXW7 interaction. Accordingly, p.Thr240Pro and p.Thr239Pro/p.Thr240Pro but not p.Thr239Pro blocked FBXW7-induced SOX9 degradation in cultured cells. All variants increased SOX9-mediated reporter activation independently of protein stabilization, suggesting that CPD may also modulate the transactivation function of SOX9. Altogether, these findings concur that CPD has critical functions, that SOX9 decisively controls odontogenesis, and that gain-of-function variants may markedly perturb both this process and skeletogenesis.

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.

To prospectively evaluate the prognosis of fetuses diagnosed with micrognathia using prenatal ultrasound screening. Between January 2019 and December 2022, a normal range of IFA to evaluate the facial profile in fetuses with micrognathia in a Chinese population between 11 and 20 gestational weeks was established, and the pregnancy outcomes of fetal micrognathia were described. The medical records of these pregnancies were collected, including family history, maternal demographics, sonographic findings, genetic testing results, and pregnancy outcomes. Ultrasound identified 25 patients with fetal micrognathia, with a mean IFA value of 43.6°. All cases of isolated fetal micrognathia in the initial scans were non-isolated in the following scans. A total of 78.9% (15/19) cases had a genetic cause confirmed, including 12 with chromosomal abnormalities and 3 with monogenic disorders. Monogenic disorders were all known causes of micrognathia, including two cases of campomelic dysplasia affected by SOX9 mutations and one case of mandibulofacial dysostosis with an EFTUD2 mutation. In the end, 19 cases were terminated, 1 live birth was diagnosed as Pierre Robin syndrome, and 5 cases were lost to follow-up. IFA is a useful indicator and three-dimensional ultrasound is a significant support technique for fetal micrognathia prenatal diagnosis. Repeat ultrasound monitoring and genetic testing are crucial, with CMA recommended and Whole exome sequencing performed when normal arrays are reported. Isolated fetal micrognathia may be an early manifestation of monogenic disorders.

SOX9 is an essential transcriptional regulator of cartilage development and homeostasis. In humans, dysregulation of SOX9 is associated with a wide spectrum of skeletal disorders, including campomelic and acampomelic dysplasia, and scoliosis. The mechanism of how SOX9 variants contribute to the spectrum of axial skeletal disorders is not well understood. Here, we report four novel pathogenic variants of SOX9 identified in a large cohort of patients with congenital vertebral malformations. Three of these heterozygous variants are in the HMG and DIM domains, and for the first time, we report a pathogenic variant within the transactivation middle (TAM) domain of SOX9 . Probands with these variants exhibit variable skeletal dysplasia, ranging from isolated vertebral malformation to acampomelic dysplasia. We also generated a Sox9 hypomorphic mutant mouse model bearing a microdeletion within the TAM domain ( Sox9 Asp272del ). We demonstrated that disturbance of the TAM domain with missense mutation or microdeletion results in reduced protein stability but does not affect the transcriptional activity of SOX9. Homozygous Sox9 Asp272del mice exhibited axial skeletal dysplasia including kinked tails, ribcage anomalies, and scoliosis, recapitulating phenotypes observed in human, while heterozygous mutants display a milder phenotype. Analysis of primary chondrocytes and the intervertebral discs in Sox9 Asp272del mutant mice revealed dysregulation of a panel of genes with major contributions of the extracellular matrix, angiogenesis, and ossification-related processes. In summary, our work identified the first pathologic variant of SOX9 within the TAM domain and demonstrated that this variant is associated with reduced SOX9 protein stability. Our finding suggests that reduced SOX9 stability caused by variants in the TAM domain may be responsible for the milder forms of axial skeleton dysplasia in humans.